The long-term goal of this project is to understand the molecular mechanism by which proteins are transported across or are inserted into the mammalian endoplasmic reticulum (ER) membrane. We have recently established a system of reconstituted proteoliposomes which reproduces the translocation process with purified membrane proteins and pure phospholipids. The minimum translocation apparatus of the ER membrane seems to comprise only the Sec61p- complex of three subunits and the TRAM protein. We now propose to thoroughly analyze the mechanism of translocation, in terms of these individual components. Specifically, we will use crosslinking and reconstitution methods to address the following questions: 1. What are the steps of translocation on a molecular level? 2. What are the functions of the Sec61p- complex? 3. How does the TRAM protein facilitate translocation? We will also search for additional components which may be required for the postranslational mode of translocation, for the recycling of translocation components and for the regulation of the translocation process. We believe that our studies will contribute significantly to the understanding of the first and decisive step in the biosynthesis of a large class of proteins which includes secretory proteins, proteins of the plasma membrane, of lysosomes and of all organelles of the secretory pathway.